The present invention relates to a substantially continuous enzymatic process for preparing optionally N-protected peptides, especially dipeptides, and to an apparatus for use therefor
It is possible that di- and oligopeptides such as, for example, tyr-ala will in the future play an important part in parenteral nutrition: they cause a low osmolarity and are excellently water-soluble and thus can readily be used for infusion solutions. Furthermore, some peptides are distinguished by highly interesting pharmacological properties, such as, for example, the peptide hormones (for example tyr-arg). However, approval thereof as pharmaceutical agents requires high purity, especially enantiomeric purity above 99.9 ee. Such high enantiomeric purities can be achieved in conventional chemical processes only with great elaboration. Hence, enzymatic peptide synthesis
Johansen et al., U.S. Pat. No. 4,339,534, the entire disclosure of which is incorporated herein by reference, discloses a process for the preparation of N-protected (N-acylated) dipeptides by enzymatic reaction of N-acylamino acid alkyl esters (A) with optionally C-protected amino acids (B) using carboxypeptidase as the enzyme, which can be in solution, insolubilized or immobilized on a carrier.
The examples in the Johansen patent reveal the utility of an organic solubilizer, e.g., an alcohol, in some cases, and the use of a large excess of amino acid relative to the ester. In addition, the reported results show a distinct improvement when the amino acid component B is used in the form of the amide and not as the free acid (which, however, makes an additional protective group removal necessary in order to obtain the peptide itself).
Detailed information on the elimination of the protective groups, especially of the N-acyl group, from the N-protected dipeptide is not provided in the Johansen patent.
The examples disclose a batchwise preparation with chromatographic fractionation of the reaction mixture at the end.
Further details of a procedure for the preparation of N-acyl-dipeptide amide taking the example of the reaction of N-acyl-arginine with methioninamide in the presence of immobilized carboxypeptidase (CPD) are given by Cramer et al., in J. Chromat., 394:305-314 (1987), who describe peptide synthesis in a fixed bed reactor with recycled reaction mixture and working-up of the product after sufficient reaction by displacement chromatography of the reaction mixture, which has been adjusted to pH 2.5 in an intermediate container, in batch operation.
The N-protective groups which are mainly exemplified in the abovementioned Johansen patent are benzoyl, acetyl and benzyloxycarbonyl from a plurality of groups stated to be possible. However, Widmer et al. ("Enzymatische Peptidsynthese" in "Peptides 1982" Walter de Gruyter & Co. Berlin New York, 1983, pages 375 to 379) disclose the use of the N-phenacetyl group as a protective group for the reaction of N-acyl-amino acid alkyl esters with amino acid amides in the presence of carboxypeptidase-Y (CPDY), and they indicate that it can be expected that the N-phenacetyl group could be enzymatically cleaved from small peptides using penicillin acylase.
Fuganti et al., Tetrahedron Letters, 27:3191-3194 (1986), disclose successful enzymatic deacylation of N-phenacetyl L,L-aspartame (N-phenacetyl L,L-aspartyl-phenylalanine methyl ester), a C-protected (esterified) N-phenacyldipeptide, using penicillin acylase immobilized on Eupergit. The phenacyl group was similarly but more slowly cleaved from N-phenacetyl L,L-aspartyl-valine methyl ester while the protective group was not eliminated from the N-acylated aspartic acid itself
A need continues to exist for an economical and efficient process for synthesizing dipeptides and oligopeptides.